Trichinosis is a disease of world-wide distribution that is primarily due to the ingestion of raw or undercooked meat (principally pork) containing the infective larval stage of Trichinella spiralis, the helminth parasite that causes the disease. After ingestion, Trichinella spiralis larvae infect the intestine where they mature within a few days. Female worms then bear newborn larvae which enter the general circulatory system and after several days accumulate in the striated muscles of the infected animal. Until recently, Trichinella spiralis infection has been detected by visual detection of larvae in muscle snips or digestion of muscle to liberate larvae (see, for example, U.S. Pat. No. 3,892,529 by Giles, issued Jul 1, 1975, and U.S. Pat. No. 3,918,818 by Giles, issued Nov. 11, 1975).
A group of protein (including glycoprotein) antigens extracted from Trichinella spiralis muscle stage larvae has been the subject of numerous studies in recent years, particularly in attempts to develop vaccines and diagnostic agents for trichinosis. These larval antigens are highly immunodominant and are apparently only present during the first muscle larval stage (L.sub.1) of Trichinella spiralis infection, being found on both the cuticular surface and excretory/secretory (ES) products of L.sub.1 larvae (see, for example, Denkers et al., pp. 241-250, 1990, Mol. Biochem. Parasitol., Vol. 41). These larval antigens, designated TSL-1 antigens by Appleton et al., pp. 190-192, 1991, Parasitol. Today, Vol. 7, evoke a strong IgG.sub.1 antibody response in mice following oral infection (see, for example, Denkers et al., pp. 3152-3159, 1990, J. Immunol., Vol. 144) and induce substantial protection against challenge infections (see, for example, Silberstein et al., pp. 898-904, 1984, J. Immunol., Vol. 132; Silberstein et al., pp. 516-517, 1985, J. Parasitol., Vol. 71; Gamble, pp. 398-404, 1985, Exp. Parasitol., Vol. 59; Gamble et al., pp. 2396-2399, 1986, Am. J. Vet. Res., Vol. 47; Ortega-Pierres et al., pp. 563-567, 1989, Parasitol. Res., Vol. 75; Denkers et al., J. Immunol., ibid.; Jarvis et al., pp. 498-501, 1992, Parasite Immunol., Vol. 14).
TSL-1 antigens migrate under reducing conditions on SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) in a molecular weight range of 40-70 kilodaltons (kDa). Denkers et al., Mol. Biochem. Parasitol., ibid., have shown that at least six of the TSL-1 antigens share a common, highly immunodominant determinant. Use of monoclonal antibodies raised against the determinants indicated that the determinants are quite selective for Trichinella spiralis in that the monoclonal antibodies did not recognize other parasites, including the closely related species Trichuris muris (Denkers et al., pp. 403-410, 1991, Exp. Parasitol., Vol. 72). Moreover, the determinants appear to be ubiquitous among all Trichinella spiralis isolates tested so far (see, for example, Denkers et al., Exp. Parasitol., ibid.; Gamble et al., pp. 67-74, 1984, Am. J. Vet. Res., Vol. 46; Gamble et al., pp. 379-389, 1984, Vet. Immunol. Immunopath., Vol. 6).
TSL-1 antigens are believed to have both protein and carbohydrate immunoreactive determinants, although there is some conflict in the literature about the importance of each. Denkers, et al., Mol. Biochem. Parasitol., ibid., demonstrated that the immunodominant determinants can be removed by treatment with trifluoromethanesulfonic acid, mild base, or N-glycanase, suggesting that the determinants are associated with both N-linked and O-linked carbohydrate groups. Denkers et al. also showed that the immunodominant determinants were not phosphorylcholine but did not further identify the composition of the carbohydrate moiety. Gold et al., pp. 187-196, Mol. Biochem. Parasitol., Vol. 41, isolated TSL-1 antigens of 43 kDa and 45-50 kDa and treated them with N-glycanase. The deglycosylated antigens were no longer able to bind to polyclonal antibodies raised against the glycosylated versions of the proteins, again suggesting the importance of carbohydrate moieties, although Gold et al. do not exclude the possibility of peptide epitopes as well.
In contrast, Jarvis et al., ibid., concluded that protein epitopes alone could induce protective immunity to Trichinella spiralis, having shown that ES antigens that had been deglycosylated using sodium periodate were as effective as native ES antigens in protecting mice from Trichinella spiralis infection in both active and passive immune assays. Su et al., pp. 331-336, 1991, Mol. Biochem. Parasitol., Vol. 45, reported that a recombinant fusion protein consisting of beta-galactosidase joined to the 49-kDa TSL-1 antigen (P49) produced in Escherichia coli was bound by antibodies contained in serum isolated from swine infected with Trichinella spiralis, and by antibodies contained in serum isolated from mice immunized with native P49 antigen, but was not recognized by three monoclonal antibodies that bind selectively to native P49 antigen. Su et al. concluded that such results suggest that, at least the P49 antigen has both protein and carbohydrate immunoreactive determinants.
Several investigators have developed enzyme-linked immunosorbent assays (ELISAs) to detect Trichinella spiralis infection using a variety of reagents, such as, crude Trichinella spiralis parasite preparations, partially purified ES antigen preparations, and monoclonal antibodies raised against, for example, the ES immunodominant determinants (see, for example, Ruitenberg et al., pp. 67-83, 1976, J. Immunol. Methods, Vol. 10; Gamble et al., 1983, pp. 349-361, Vet. Parasitol., Vol. 13; Gamble et al., Am. J. Vet. Res., ibid.; Gamble et al., Vet. Immunol. Immunopath., ibid.; U.S. Pat. No. 4,670,384, by Gamble et al., issued Jun. 2, 1987). Assays based on undefined crude or semi-defined antigen preparations are problematic due to false-positive and false-negative reactions as well as to cross-reactivity with antibodies corresponding to antigens of other parasites. Monoclonal antibody-based or purified protein-based assays, while often leading to fewer false-positive or false-negative reactions, can still have specificity and selectivity problems, in addition to difficulties of producing such components without batch-to-batch variation, and of maintaining the stability of the components.
U.S. Pat. No. 4,795,633, by Murrell et al., issued Jan. 3, 1989, discloses a swine trichinosis vaccine consisting of an inert newborn larvae preparation emulsified with an adjuvant. GB 1,580,539, published Dec. 3, 1989, discloses a trichinosis vaccine containing ES antigens of Trichinella spiralis muscle stage larvae. Several groups of investigators have reported the cloning of at least portions of certain Trichinella spiralis antigen genes with the goal of developing defined diagnostics reagents and/or vaccines (see, for example, Su et al., Mol. Biochem. Parasitol., ibid.; Sugane et al., pp. 1-8, 1990, J. Helminth., Vol. 64; Zarlenga et al., pp. 165-174, 1990, Mol. Biochem. Parasitol., Vol. 42). Problems with protein-based vaccines, and particularly with recombinant protein-based vaccines, include difficulty of preparation (particularly with respect to removal of harmful contaminants), lack of stability, potential reduced antigenicity compared to the native protein, and potential autoimmune reactions due to similarities between parasite and animal host proteins (e.g., Brugia pahangi glutathione peroxidase and Dirofilaria immitis superoxide dismutase; see, for example, Callahan et al., 235-252, 1991, Mol. Biochem. Parasitol., Vol. 49).
A number of anthelminthic drugs have been developed to treat trichinosis (see, for example, U.S. Pat. No. 5,140,042 by Arison et al., issued Aug. 18, 1992; U.S. Pat. No. 5,089,530 by Tsipouras et al., issued Feb. 18, 1992; U.S. Pat. No. 5,073,567, by Maeda et al., issued Dec. 17, 1991; U.S. Pat. No. 5,008,250, by Fisher et al., issued Apr. 16, 1991; and U.S. Pat. No. 4,833,168, by Wyvratt, issued May 23, 1989). Such drugs, however, apparently cannot be used to prevent trichinosis, are expensive to produce, usually have undesirable side effects, and are not always effective.
There remains a need for both diagnostic reagents to detect Trichinella spiralis infection and for vaccines and other drugs to protect animals from trichinosis that have improved specificity, selectivity, stability, consistency, and ease of use.